Cornea, a transparent tissue comprising five cellular layers, i.e. corneal epithelium, Bowman membrane, corneal stromal layer, Descemet's membrane, and corneal endothelium from outside to inside in the order, contains no blood vessel but nerves. Corneal epithelium is the non-keratinized stratified squamous epithelium having a thickness of about 50 μm and constitutes a part of the ocular surface. It functions as a barrier which limits the penetration of substances and prevents the invasion of pathogen such as bacteria and fungi to the inside of cornea. Tight junctions existing between adjacent cells in the outermost layer play an important role in maintaining the barrier functions of the corneal epithelium.
Corneal epithelium has a regeneration ability. Even if epithelium is injured, corneal epithelial cells are supplied by corneal epithelial stem cells and then tight junctions between adjacent cells are regenerated, resulting in the recovery and the maintenance of the barrier functions.
Keratoplasty is prevalent as an effective treatment technique for a corneal disease caused by the irreversible impairment of the barrier functions of the corneal epithelium. There are two kinds of keratoplasties; one is a penetrating keratoplasty in which full-thickness cornea is transplanted, and the other is a keratoplasty in which part of the corneal tissue is transplanted. Because the penetrating keratoplasty has a problem such as vulnerability of eyeball caused by dissection of all the layers of cornea, the keratoplasty in which part of the corneal tissue is transplanted has been prevalent in recent years. As the partial corneal tissue transfer noted above, there are two kinds of surgical techniques, one is a lamellar keratoplasty in which part of the corneal stroma and corneal epithelium are simultaneously transplanted, and the other is an epithelium keratoplasty in which only corneal epithelium is transplanted. These techniques have already been established in clinical practice for patients with recurrent corneal dystrophy and the like.
Corneal epithelial stem cells differentiable into corneal epithelial cells exist in the fundus of the corneal limbal tissue. Therefore, if the corneal limbal tissue is injured, corneal epithelial stem cells are not supplied to repair corneal epithelial cells. Furthermore, when corneal epithelium becomes extensively damaged, it takes long to supply the needed volume of corneal epithelial cells from corneal epithelial stem cells to repair the damage. In addition, when Bowman's membrane which is extracellular matrix of corneal epithelial cells becomes damaged, corneal epithelial cells develop adhesive imperfection resulting in an insufficient volume of the cells on the corneal epithelium. Thus, when the condition in which barrier functions of the corneal epithelium are damaged is continuing due to the corneal epithelium being left unrepaired for several weeks, the ocular surface easily becomes infected with bacteria. Upon ocular surface being infected, the ocular surface is overlaid with conjunctival epithelium accompanying neovascularization, and inflammation occurs due to a migration of immune cells. Furthermore, corneal tissues are destroyed with enzymes secreted by bacteria, resulting in possible loss of vision. Thus, a disease caused by impairment or decrease of supply of corneal epithelial stem cells refers to corneal epithelial stem cell deficiency. Transplantation of corneal epithelium to cornea developing inflammation caused by corneal epithelial stem cell deficiency shows decreased survival rate due to the rejection.
Furthermore, corneal epithelium transplantation has the problem of donor shortage in which enough corneal epithelium to meet needs cannot be obtained for use in the transplantation. To overcome the donor shortage, a method for transplantation in which cell sheets obtained by culturing corneal epithelial cells in-vitro are transplanted onto the damaged area of the corneal epithelium has been proposed. In such a case where the human corneal epithelial sheets are transplanted onto the damaged area, inflammation at the site of transplantation also becomes a problem. Therefore, a method to transplant human corneal epithelial sheet with the amnion layer has been tried to suppress the inflammation, in which the sheet was formed on the amnion.
A method to form cell sheet on the amnion has been reported where pieces of corneal epithelial limbus taken from a healthy white rabbit were seeded on an amnion substrate (amnion from which amnion epithelial cells were stripped off and removed), and then were co-cultured with feeder cells (NIH-3T3 cells) (Non Patent Literature 1). It has been confirmed that human corneal epithelial sheet obtained as above had a layered structure morphologically similar to living corneal epithelium. Based on the clinical trial in which human corneal epithelial sheets have been transplanted to patients with refractory ocular-surface disorders, wherein the sheet was formed by a similar method using pieces of healthy human corneal epithelial limbus, it has showed that transplantation of the corneal epithelial sheet obtained as above was effective as a method to recover eye functions (Non Patent Literatures 2 and 3).
The other method to form cell sheets on the amnion has been reported where corneal epithelial cells which were obtained by enzymatically treating pieces of corneal epithelial limbus taken from a healthy white rabbit or human were suspended and seeded on an amnion substrate (amnion from which amnion epithelial cells were stripped off and removed), and then were co-cultured with feeder cells (NIH-3T3 cells) (Patent Literature 1 and Non Patent Literature 4). It also has been confirmed that human corneal epithelial sheet obtained as above had a layered structure morphologically similar to living corneal epithelium. In addition, induction of tight junction-related protein expression were confirmed by culturing cells whereby the cell surface was exposed to the atmosphere (air-lifting) during the culture process of human corneal epithelial sheets.